Quantitative Fluorescence

The phenomenon of fluorescence has revolutionized research in biology and medicine. More than 100 years ago, Paul Ehrlich began to develop specific chemical staining methods and the latest development of light-controlled proteins (optogenetics) will surely not mark the end of this progression. A historically continuous trend is the quest for measuring and quantifying data, with or without generation of images as data source. Many techniques have been introduced that are based on fluorescence, but not necessarily require image formation. The laboratory jargon refers to this methods as “F-techniques”: FLIM, FCS, FCCS, FRET, FRAP… to mention just the most important. Read the articles in this topic, to find out more details on these modern methods.

Only a few days to go before the start of Focus on Microscopy 2015 in Göttingen, Germany. This year’s FOM is being organized by Dr. Gabriele Bunt and Prof. Dr. Fred S. Wouters from the University Medical Center, Göttingen, in cooperation with Prof. Dr. G.J. (Fred) Brakenhoff, University of Amsterdam, The Netherlands.

Confocal microscopy has come a very long way since its invention more than a half-century ago. Today, with novel technology driven by leading imaging companies, it has become the standard for fluorescence microscopy. Choosing the right confocal microscope for your specific research requires the appropriate mix of features related to resolution, sensitivity, and speed.

To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves.

Small brightly fluorescent carbon nanoparticles have emerged as a new class of materials important for sensing and imaging applications. We analyze comparatively the properties of nanodiamonds, graphene and graphene oxide ‘dots’, of modified carbon nanotubes and of diverse carbon nanoparticles known as ‘C-dots’ obtained by different methods.

Fluorescence recovery after photobleaching (FRAP) has been considered the most widely applied method for observing translational diffusion processes of macromolecules. State of the art laser scanning microscopes such as the Leica TCS SP8 have the advantage of using a high intensity laser for bleaching and a low intensity laser for image recording. The LAS AF application wizard offers different ways to carry out a FRAP experiment.

For the understanding of functions of proteins in biological and pathological processes, reporter molecules such as fluorescent proteins have become indispensable tools for visualizing the location of these proteins in intact animals, tissues, and cells. For enzymes, imaging their activity also provides information on their function or functions, which does not necessarily correlate with their location. Metabolic mapping enables imaging of activity of enzymes.

In eukaryotic cells Ca2+ is one of the most widespread second messengers used in signal transduction pathways. Intracellular levels of Ca2+ are usually kept low, as Ca2+ often forms insoluble complexes with phosphorylated and carboxylated compounds. Typically cytosolic Ca2+ concentrations are in the range of 100 nM. In response to stimuli Ca2+ may either be released from external medium or internal stores to raise the Ca2+ concentration.

Imaging techniques are indispensable in many fields of life sciences today. With state-of-the-art optics and metrology, they provide hundreds of gigabytes of still images and videos. Correspondingly, there is a growing need for complex software solutions to ensure that the amounts of generated data can be automatically managed, processed and analyzed – and shared online with a large group of users. The combination of individual open-source software projects is proving especially useful for solving such complex image analysis problems.

In this article, advantages and disadvantages of different types of sensors for single point true confocal scanning devices are discussed. Traditionally, photomultiplier tubes have been employed in such systems. For some cases, avalanche photodiodes have proven to fit best. A new development uniting vacuum and silicon technology has led to chimeric sensors, called hybrid detectors (HyD). They benefit from both technologies.

FLIM combines lifetime measurements with imaging: lifetimes obtained for each image pixel are color-coded to produce additional image contrast. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its biochemical status or nano-environment. A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well.

Fluorescence correlation spectroscopy (FCS) measures fluctuations of fluorescence intensity in a sub-femtolitre volume to detect such parameters as the diffusion time, number of molecules or dark states of fluorescently labeled molecules. The technique was independently developed by Watt Webb and Rudolf Rigler during the early 1970s.